Bovine Somatotropin (BSt) is a growth hormone that has been well studied and the literature has been recently reviewed by Paladini, A. C. et al., CRC Crit. Rev. Biochem., 15:25-56 (1983). Somatotropins were originally discovered in pituitary gland extracts from various animal species. In general, somatotropins are conserved molecules and similarities in amino acid sequences and structure are found between animals of disparate evolutionary ranking.
Growth hormones including bovine growth hormone are globular proteins comprised of a single chain of approximately 200 amino acids, having 2-3 intramolecular disulfide bonds. Specifically, BSt has a single chain of 190-191 amino acids, a globular structure with two intramolecular disulfide bonds, and a molecular weight of approximately 22,000 daltons.
BSt extracted from pituitary glands is heterogeneous. At least six major forms of the protein have been described. The longest form has 191 amino acid residues and an ala-phe amino terminus. The second form has 190 amino acid residues and a phe amino terminus. The third form has 187 amino acid residues and a met amino terminus. The remaining three forms of BSt substitute valine for leucine at position 127. In addition to this heterogeneity, undefined heterogeneity of bovine somatotropin has also been described (Hart, I. C. et al., Biochem. J., 218:573-581 (1984); Wallace, M. and Dickson, H. B. F., Biochem. J., 100:593-600 (1965)).
Undefined electrophoretic heterogeneity is seen when the native extracts are fractionated by anion exchange chromatography. It has not been shown that the defined forms have different relative potency in bioassays. However, it has been shown that the undefined species of BSt when fractionated on ion exchange columns demonstrate varying degrees of bioactivity in rat growth models (Hart, et al. and Wallace and Dickson, supra.).
It is not known whether undefined heterogeneity exhibiting biological variation is due to genetic variability, to in vivo post-translational modification, to differences in phosphorylation (Liberti, J. P. et al., Biochem. and Biophys. Res. Comm., 128:713-720, (1985)), or to artifacts of isolation.
BSt either produced by recombinant microorganisms or extracted from pituitary gland tissue is estimated to be of great commercial value. It has the ability to increase lactation in dairy cattle and has the ability to increase size and meat production in beef cattle. Administration to cattle is a problem. It is estimated that upwards to 100 mg per animal per day or more will be needed to effect commercially acceptable improvements in production. Such a dosage will require efficient methods of administration. Improvements in the potency and stability of BSt such as described in this invention will be of benefit because of resulting reductions in the amount of drug administered to each animal per day.
Furthermore, one of the problems in preparing recombinantly-produced BSt (rBSt) is that liquid processing and storage of rBSt at acid or alkaline pH results in the conversion of the asparagine residue at position 99 to isoaspartic acid. The resulting rBSt is referred to as "early eluting rBSt" because it elutes earlier than native rBSt on reversed phase HPLC. Isoaspartate is formed when the asparagine side chain condenses with the peptide backbone resulting in the elimination of ammonia. Chain cleavage also occurs by a condensation reaction between the peptide backbone and the asparagine residue at position 99 of the rBSt molecule upon storage. The chain-cleaved product is covalently held together by the disulfide bond between cysteine residues 53 and 164 and has been called "early-early eluting rBSt" because of its eluting position relative to native and early eluting rBSt.
Because the instability that occurs due to the modification of asparagine 99 leads to a loss of native rBSt during its isolation, formulation and storage as reconstituted product, it would be advantageous to make an amino acid substitution at position 99 to produce an rBSt analog that is more stable while retaining or enhancing its biological activity.